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The anti-proliferative effect of different tomato varieties on the human colon adenocarcinoma cells

The anti-proliferative effect of different tomato varieties on the human colon adenocarcinoma cells Volume 2 † Number 2 † June 2009 10.1093/biohorizons/hzp020 ......................................................................................................................................................................................................................................... Research article The anti-proliferative effect of different tomato varieties on the human colon adenocarcinoma cells Caroline Saunders* Department of Food Biosciences, School Chemistry, Food and Pharmacy, University of Reading, Reading, UK. * Corresponding author: 47 Beech Lane, Earley, Reading, Berkshire RG6 5PT, UK. Tel: þ44 1189 671022. Email: caroline.saunders50@ntlworld.com Supervisor: Dr Jeremy P.E. Spencer, Molecular Nutrition Group, School Chemistry, Food and Pharmacy, University of Reading, Reading, UK ........................................................................................................................................................................................................................................ Epidemiological evidence suggests that diets rich in fruit and vegetables protect against the development of colon cancer, primarily due to their high levels of polyphenols, in particular a group known as flavonoids. Tomatoes contain significant amounts of the glycosides of the flavonoids quercetin and naringenin. Both quercetin and naringenin have been shown to exert anti-proliferative effects on colon cancer cells, although the effects of whole tomato polyphenol extracts are unclear. The aim of this study was to determine the effect of three tomato varieties, with differing levels of flavonoids and total phenolics, on the proliferation of human colon adenocarci- noma cells. We show that all three varieties were able to significantly inhibit the growth of colon cancer cells, although this activity was found not to be linked to the levels of the flavonoids rutin and naringenin in the tomatoes, but rather to their total phenolic content. We show that the mechanism of growth inhibition was linked to the effects of tomato phenolics on the cell cycle, in that exposure of cells to the tomato extract induced a reduction in the percentage of cells in the S-phase, i.e. those actively synthesizing DNA. These data indicate that tomatoes may help to prevent colon cancer but that their flavonoid content does not appear to determine the magnitude of their biological effect. Key words: colon cancer, tomato, flavonoids, naringenin, rutin. ........................................................................................................................................................................................................................................ and vegetables appear to provide protection against cancer Introduction development due to their levels of fibre and their high con- Colorectal cancer is one of the major cancers of the Western tents of phytochemicals, in particular flavonoids. world with countries such as Australia and New Zealand Flavonoids are secondary plant metabolites, which are having a 20-fold higher incidence than countries in Middle synthesized from phenylalanine via the Shikimate Pathway, Africa. The two major risk factors in colon cancer aetiology and express many functions within the plant, including regu- have been proposed, genetic disposition and diet, although lation of growth and UV protection. The basic structure of the variability between countries appears to result from the flavonoids consists of two benzene rings connected by an latter as migrant populations develop a similar colon oxygen-containing pyrene ring (Fig. 1). On the basis of vari- cancer incidence to that of their new country within a gener- ations in the saturation of the basic flavan ring system, their ation. Indeed, epidemiological evidence recorded between alkylation and/or glycosylation and the hydroxylation 1966 and 1996 has suggested very strong links between pattern of the molecules, flavonoids may be divided into diet and cancer of the breast, colorectum and prostate. seven subclasses: flavonols, flavones, flavanones, flavanonols, One of the reasons that the colon is so susceptible is due to flavanols, anthocyanidins and isoflavones (Fig. 1). Evidence its direct exposure to dietary carcinogens such as N-nitroso indicates that flavonoids express a wide variety of biological 7 6 compounds, such as those present in preserved meats, activities including antioxidant nature, anti-inflammatory which cause DNA damage and lead to mutations and the and anti-aggregatory effects and an ability to inhibit 3 9–12 initiation of carcinogenesis. In contrast, diets high in fruit cancer cell growth. Tomatoes (Lycopersicon ......................................................................................................................................................................................................................................... 2009 The Author(s). This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. 172 Bioscience Horizons † Volume 2 † Number 2 † June 2009 Research article ......................................................................................................................................................................................................................................... Figure 1. Basic structure of flavonoids consisting of two benzene rings connected by an oxygen containing pyrene ring. Sub-classes of flavonoids: flavonols, flavanones, flavanols, anthocyanidins and with differing R groups that give each flavonoid its unique structure. esculentum) are one of the world’s common vegetables and which is produced from naringenin chalcone during tomato contain significant amounts of the flavonol, rutin (quercetin processing. 3-rutinoside) and the flavanone, naringenin and naringenin A number of case–control studies have found an inverse 13, 14 chalcone. Rutin is present in tomato as a glycoside relationship between tomato intake and the risk of develop- 18, 19 and as such undergoes very limited absorption in the small ing colorectal cancer. Furthermore, in vitro and in vivo intestine, as the b-glycosidic bond of the rutinose moiety is studies have demonstrated a wide range of biological activi- resistant to hydrolysis by digestive enzymes. However, ties related to tomato flavonoids, with quercetin and rutin enzymes produced by the colonic gut microflora are able to inhibit abnormal colonic growths in rats and quer- capable of breaking this glycosidic linkage to yield quercetin, cetin chalcone, capable of reducing the size of implanted 9, 16 21 which has been shown to be bioavailable. Flavonoids colon tumours in mice. In humans, quercetin, in combi- that are not well absorbed in the small intestine and the nation with the polyphenol curcumin, was shown to reduce major proportion of ingested flavonoids pass to the large the number and size of ileal and rectal adenomas in patients intestine where they may express biological function on with familial adenomatous polyposis (FAP). Furthermore, host cells and bacteria. This is also true of naringenin quercetin, but not rutin, has been shown to express ......................................................................................................................................................................................................................................... 173 Research article Bioscience Horizons † Volume 2 † Number 2 † June 2009 ......................................................................................................................................................................................................................................... anti-proliferative effects on human colon cancer cell lines, in including a sample selectively bred to be high in polyphenols, 9, 10, 23, 24 a dose-dependent and time-dependent manner. a low flavonoid mutant and a traditional Ailsa Craig variety. 9, 23 Their ability to induce apoptosis and/or evoke cell All samples were stored in the dark at room temperature. 25 –27 cycle arrest have been proposed as potential anti-cancer Polyphenols, hydroxycinnamates, flavonoids and their glyco- mechanisms. The latter has been proposed to occur via their sides were extracted from lyophilized tomato by three ability to regulate cyclins, a family of proteins that are posi- sequential aqueous/methanol extractions. A methanol/ tive regulators of the cell cycle and which are commonly water (2:1) solution was prepared and a total volume of over-expressed in cancer. In response to growth factors 1 ml added to 100 mg lyophilized tomato. Samples were vor- and mitogens, cyclins associate with cyclin-dependent texed continuously for 30 min using a vibrating agitator. kinases (CDKs), which then phosphorylate cellular proteins Samples were then microfuged at 10 000 rpm for 5 min at resulting in the progression of the cell through the cell cycle. 4 C and the supernatants collected. The pellet was A tomato digestate containing both rutin and naringenin re-suspended in 1 ml of the aqueous methanol solution and chalcone has been shown to arrest cell cycle progression at the procedure repeated a further two times. Samples were the G0/G1 phase in colon adenocarcinoma cells through purged with argon and protected from light throughout the decreased expression of cyclin D1, a positive regulator of extraction. the cell cycle. Furthermore, many flavonoids present in tomatoes have been shown to be capable of inhibiting the Quantification of Total Phenolics cell cycle and modulating cyclin expression. For example, The total phenolic content of the aqueous methanolic extract 2 -OH flavanone, a flavonoid with structural similarities to of each tomato sample was obtained using the Folin– naringenin, induces cytotoxic effects on three colorectal car- Ciocalteau Micro method as previously described. cinoma cells via a mechanism dependent on increased Twenty microlitres of the tomato extracts (10 mg/ml) were expression of p21, a potent CDK inhibitor capable of block- mixed with 1.58 ml water and 100 ml of the Folin– ing cells in the G1/S phase of the cell cycle. In addition, Ciocalteu reagent, vortexed for 5 min. Following this, quercetin has been shown to block the cell cycle at the 300 ml of sodium carbonate solution (250 mg/ml) was G1/S phase in human colon cancer cells, by inhibiting the added and the reactants were mixed and left at 4 C for synthesis of a 17 kDa protein. In this study, we have deter- 30 min. Absorbance was determined at 765 nm against the mined the major flavonoid content and total polyphenol blank, and a gallic acid calibration curve (0–500 mg/l) levels of three different tomato varieties and related this to was constructed and used to determine the total phenolic their ability to inhibit the growth of human epithelial color- content of the samples, expressed as Gallic Acid ectal adenocarcinoma cells. In addition, we have attempted Equivalents (GAE). to determine how they act via assessment of their capability to induce cell cycle arrest. Assessment of Flavonoid Levels Reverse phase HPLC (RP-HPLC) was performed to charac- Materials and Methods terize and quantify the flavonoids rutin and naringenin. A Hewlett Packard (Agilent, Bracknell, UK) model 1100 Chemicals and Reagents series LC running HP ChemStation software with a Nova Human epithelial colorectal adenocarcinoma cells (Caco-2) Pak C column (250  4.6; 4 mm) (Waters, Elstree, UK) were obtained from ECACC (Salisbury, Wiltshire, UK). was used to separate the phenolic constituents. The mobile Methanol [high-performance liquid chromatography phase consisted of (A) aqueous methanol (5%) containing (HPLC) grade] and N, N-dimethylformamide (analytical HCl (0.1%) and (B) acetonitrile (50%) containing HCl grade) were purchased from Fisher (Loughborough, UK); (0.1%) and was pumped through the column at 1 ml/min Anti-bromodeoxyuridine (BrdU) Pure was purchased from with an injection volume of 50 ml. The following gradient Becton Dickinson (Oxford, UK); DMEM was purchased system was used over a time course of 60 min (%A/%B): from Cambrex Bio Science (Wokingham, UK). All other 95/5, 50/50, 0/100 and 95/5. HPLC profiles of extracts cell culture components were from Gibco Invitrogen were measured by a diode-array detector set at four wave- (Paisley, UK). All other chemicals were from Sigma-Aldrich lengths: 254, 280, 320 and 365 nm and fluorescence detec- (Gillingham, UK). tion at excitation 276 nm, emission 316 nm. Flavonoids were identified by matching their retention times and UV Tomato Samples and Extraction diode array spectra with those of authentic compounds. Lyophilized tomato samples were supplied by Professor Peter Calibration curves of rutin and naringenin were constructed Bramley, School of Biological Sciences, Royal Holloway, using authentic standards (0–100 mM) and in each case were University of London. All samples were glasshouse grown found to be linear with correlation coefficients of 0.998 under supplementary lighting. Three samples were used, (rutin) and 0.999 (naringenin). ......................................................................................................................................................................................................................................... 174 Bioscience Horizons † Volume 2 † Number 2 † June 2009 Research article ......................................................................................................................................................................................................................................... Cell Culture, Treatment and Assessment of Proliferation Statistics Caco-2 cells were grown as monolayers in T-75 culture flasks All data are expressed as means+ SD. The results of the (Greiner-Bio-One, Frickenhausen, Germany). Cells were cul- Folin–Ciocalteu assay were subjected to a one-way tured in Dulbecco’s modified Eagle’s medium, supplemented ANOVA with Tukey’s post hoc test. Statistical analysis of with 20% heat-inactivated foetal bovine serum, 2 mM Caco-2 proliferation in response to tomato treatments was L-glutamine, 1% non-essential amino acids, 100 U/ml peni- performed using a two-way ANOVA with Dunnet’s post cillin and 100 mg/ml streptomycin and stored in a humidified hoc test. Differences were considered significant when p , 37 C incubator with 5% carbon dioxide. Medium was 0.05. All statistical analyses were performed using SPSS. changed every 2 days and cells were passaged every 7 days using trypsin-versene (EDTA). Prior to the addition of tomato extracts, cells were seeded in 12-well plates (1.2  Results 10 cells/well). When cells were 30% confluent, they were Polyphenolic Content of the Tomato Extracts exposed to the tomato extracts (0.33–3.3 mg). Following 24, 48, 72 and 96 h, cells were fixed and cell biomass was Quantification of phenolics was determined by the Folin– determined using the sulforhodamine B (SRB) assay, as Ciocalteu Assay and statistical analysis using a one-way described previously. Cells were fixed by the addition of ANOVA with Tukey’s post hoc test. Results were expressed 125 ml ice-cold TCA (10% final concentration; 48C; 1 h). as GAE and indicated that the Ailsa Craig variety had a sig- After fixing, media was removed, cells were washed and nificantly higher phenolic content ( p , 0.001) compared total biomass was determined using SRB (250 ml of 0.4% with the selectively bred high-polyphenol variety and the SRB; 0.5 h). Unincorporated dye was discarded by washing low-polyphenol mutant (Fig. 2). However, the high- with 1% acetic acid, while cell incorporated dye was solubil- polyphenol variety had significantly higher levels of total ized using Tris Base (10 mM, pH 10.5). Dye incorporation, phenolics compared with the low-polyphenol mutant ( p , reflecting cell biomass, was measured at 492 nm, using a 0.05) (Fig. 2). GENios microplate reader (TECAN, Reading, UK). Flavonoid Identification and Quantification HPLC analysis (365 nm) indicated that all three tomato var- ieties revealed that three major flavonoids were present, as Cell Cycle Analysis 4 defined by the appearance of peaks at detectable at 31.7, Caco-2 cells were seeded in 12-well plates (1.2  10 cells/ 34.1 and 50.4 min (Fig. 3). The Ailsa Craig variety well) and grown until 30% confluent before exposure to (Fig. 3Ai) had significantly higher levels of rutin (quercetin-3- tomato extracts (1.7 mg/ml; 48 h). After 48 h of exposure, 8 rutinoside) (RT: 34.1 min) and naringenin chalcone (RT: BrdU (10 mM) was added and cells were incubated at 37 C 50.4 min) than either the low mutant (Fig. 3Aii) or high- for 30 min, prior to washing (PBS) and harvesting of cells polyphenol tomatoes (Fig. 3Aiii). The tomatoes also con- (250 ml trypsin/well). Cells were re-suspended in ice-cold 8 tained another, more polar, flavonoid with similar spectral 70% ethanol (1 ml) and stored at 4 C for 24 h. Cells were centrifuged at 2000 rpm (5 min; 4 C), the ethanol removed and the cells re-suspended in HCl (0.1 M; 10 min) in order to permealize them. After washing, cells were incubated for 60 min with a BrdU antibody (5 mg/ml) in PBS containing 0.5% Tween 20, 1% foetal calf serum (FCS). Cells were then washed (PBS) and centrifuged (2000 rpm; 10 min; 4 C) prior to removal of the supernatant and addition of FITC-conjugated rabbit anti-mouse immunoglobulin (0.1 mM) in PBS, containing 0.5% Tween 20 and 1% FCS. This was mixed and incubated in the dark for 30 min. Following a final wash step (PBS) and centrifugation (2000 rpm; 10 min; 4 C), the supernatant was removed and propidium iodide (50 mg/ml) in PBS containing RNase (10 mg/ml) was added. The number of proliferating cells Figure 2. Total phenolic content (expressed as equivalents of gallic acid) was determined by flow cytometry using a FACS Calibur of three different varieties of lyophilized tomatoes determined by the benchtop flow cytometer (Becton-Dickinson, Oxford, UK) Folin–Ciocalteu assay and statistical analysis using a one-way ANOVA. equipped with a 15 mW blue argon laser source (excitation Data are means of three technical replicates of each sample (mean+ SD; wavelength: 488 nm) and were analysed using CellFIT n ¼ 3). A significant difference in total phenolic content between samples is represented by: *p , 0.05 and ***p , 0.001. Cell-Cycle Analysis Version 2.0.2 software. ......................................................................................................................................................................................................................................... 175 Research article Bioscience Horizons † Volume 2 † Number 2 † June 2009 ......................................................................................................................................................................................................................................... Figure 3. (A) RP-HPLC chromatogram recorded at 365 nm of tomato extracts (concentration 10 mg/ml), (i) Ailsa Craig; (ii) low polyphenol and (iii) high polyphenol. (B) Content of flavonoids, rutin and naringenin derivatives, in tomatoes extracted with aqueous methanol (2:1) and expressed as mg/glyo- philized tomato. Quantified using ‘area-under-curve’ analysis relative to an internal standard and by comparison with calibration curves of rutin and naringenin. characteristics to rutin (RT: 31.7) (Fig. 3). Although we were product. In quantitative terms, the Ailsa Craig variety had unable to characterize this compound, it is likely to be either a much higher levels of both rutin and naringenin chalcone more polar glycosidic derivative of quercetin or a myricetin compared with the low mutant or high-polyphenol varieties glycoside (Fig. 1). LC-MS/MS is underway to identify this (Fig. 3B). Although our extraction and separation protocol ......................................................................................................................................................................................................................................... 176 Bioscience Horizons † Volume 2 † Number 2 † June 2009 Research article ......................................................................................................................................................................................................................................... is capable of revealing the hydroxycinnamates, all three tomatoes were found not to contain significant levels (Fig. 3). Effect on Colon Adenocarcinoma Cell Proliferation The cytotoxic effects of the three tomato extracts were indi- cated by a reduction in cell biomass compared with vehicle- treated cells which was determined by the SRB assay. A stat- istically significant ( p , 0.05) inhibition of cell growth was apparent following exposure to Ailsa Craig (Fig. 4A) and high polyphenol (Fig. 4B) at both concentrations and at both time points and the low polyphenol (Fig. 4C) after 72 h exposure only. At the 72 h exposure time point, there was a statistically significant difference in the ability of the Ailsa Craig extract to inhibit cell proliferation compared with the low-polyphenol sample ( p , 0.05) (Fig. 5). Cells exposed to the high-polyphenol tomato extract (1.7 mg/ Figure 5. Comparison of growth inhibitory effect of the three different ml) and vehicle-treated cells were analysed by flow cytometry tomato varieties, on proliferation of Caco-2 cells, relative to vehicle-treated cells, after 72 h of exposure to sample. Values are given as means+ SD (n ¼ 4). A significant difference of growth inhibition between samples is represented by: *p , 0.05. for the number of cells actively synthesizing DNA (i.e. in the S-phase of the cell cycle) (Fig. 6). Emission spectra of FITC indicated a shift in cell cycle distribution in cells exposed to the high-polyphenol extract, as indicated by a decreased cellular incorporation of BrdU (Fig. 6). Discussion A number of case–control studies have shown an inverse relationship between tomato intake and the risk of developing 16, 17 colorectal cancer. Furthermore, in vitro and animal studies have shown that tomatoes, and phenolic compounds present in tomatoes, are able to inhibit colon cell prolifer- ation. In this study, we demonstrate that three different tomato extracts are capable of significantly inhibiting colon adenocarcinoma cell growth. However, this ability to effect growth did not appear to be dependent on the flavonoid content of the tomatoes. The most abundant flavonoids in the tomatoes we studied were the flavonol glycoside, quercetin 3-rutinoside (also known as rutin) and the flavanone, narin- 13, 14 genin chalcone. Interestingly, although the high- polyphenol variety had been selectively bred to have an increased content of polyphenols, the traditional Ailsa Craig variety had higher levels of total polyphenols and .10 times the rutin and naringenin chalcone levels than this tomato. The low-polyphenol mutant as expected had lower levels of Figure 4. Growth inhibition of Caco-2 cells induced by each methanolic total phenolics compared with both other varieties and also tomato extract treatment. Black bars: 0.5 mg/ml and grey bars: 2.5 mg/ had very low levels of rutin and undetectable levels of narin- ml, after 48 and 72 h initial exposure. (A) Ailsa Craig (B) high polyphenol genin chalcone. Although all the tomatoes induced anti- and (C) low polyphenol. All values are given as means+ SD (n ¼ 4). A sig- proliferative effects, the degree of growth inhibition (Fig. 5) nificant difference of growth inhibition at an individual time point and con- appeared to be dependent on the total phenolic content of centration, compared with vehicle-treated cells, is represented by: *p , 0.05; **p , 0.01 and ***p , 0.001. the tomatoes (Fig. 2) rather than their levels of rutin and ......................................................................................................................................................................................................................................... 177 Research article Bioscience Horizons † Volume 2 † Number 2 † June 2009 ......................................................................................................................................................................................................................................... Figure 6. Effect of high-polyphenol methanolic tomato extract on active DNA synthesis determined by the incorporation of BrdU into cell DNA during S-phase of cell cycle. Analysis was by flow cytometry. Black line: vehicle-treated cells; red line: high-polyphenol extract showing a decrease in cells in DNA synthesis phase (S-phase) of the cell cycle (n ¼ 1). naringenin (Fig. 3B). For example, the large difference in levels distribution of cells in the S-phase of the cell cycle. Cancer of rutin and naringenin in the Alisa Craig variety compared cells treated with the high-polyphenol tomato extract under- with the other two tomatoes was not reflected in their ability went a reduction in proliferation which was accompanied by to influence cancer cell growth inhibition. a reduction in the number of cells actively synthesizing DNA, This is in agreement with previous studies which suggest relative to vehicle-treated cells. This is in agreement with a that the flavonol glycoside, rutin, is not able to induce anti- previous study which showed the ability of a tomato diges- proliferative effects until the glycosidic bond of the rutinose tate to inhibit growth of HCT-116 colon adenocarcinoma moiety is cleaved to yield quercetin, which has previously cells was mediated by its ability to block the cell cycle in been shown to exert strong anti-proliferative effects. G0/G1. This block was accompanied by down-regulation Such cleavage may occur in the large intestine via the of cyclin D1, a positive regulator of the cell cycle. action of the colonic gut microflora, indicating that Furthermore, polyphenols in olive oil have been shown to although rutin is incapable of preventing the growth of inhibit proliferation of Caco-2 cells by inducing a cell cycle cancer cells in vitro, it may still be able to induce positive block in G2/M. This block was induced via the upstream reductions in the growth of cancer cells in vivo. The corre- inhibition of the MAP kinase, p38 and the transcription lation between total phenolic content and the degree of inhi- factor, CREB. Further work is required in order to determine bition of colon adenocarcinoma cell growth suggests that the precise mechanism by which tomato polyphenols induce other polyphenolic compounds, or the synergistic effect of the anti-proliferative effects in cells, although our initial a number of different polyphenols, are likely to be respon- observations suggest that they are also able to prevent cell sible for the observed growth inhibition. As our extraction cycle progression. and analysis protocol was capable of detecting and quanti- In summary, the polyphenol extracts of all three tomato fying hydroxycinnamates, our data also suggest that this varieties were able to inhibit the growth of colon adenocar- group of polyphenols is also unlikely to be responsible for cinoma cells and the growth inhibition did not appear to be the anti-proliferative effects as all the tomatoes contained due to their flavonoid or hydroxycinnamate content. The very low levels. Further experiments are necessary in order growth inhibitory effect was accompanied by a shift in cell to identify and characterize the polyphenols in the tomatoes cycle distribution, suggesting that fewer cells were present which are responsible for their biological actions on cancer in the S-phase of the cell cycle following treatment. Future cells. studies are needed in order to identify the specific com- Our data suggest that the mechanism by which the high- ponent(s) present in tomatoes which are responsible for the polyphenol tomato extract exerts its anti-proliferative growth inhibitory effects and to determine the mechanism effects is linked to its ability to induce a shift in the by which they induce their effect on the cell cycle. ......................................................................................................................................................................................................................................... 178 Bioscience Horizons † Volume 2 † Number 2 † June 2009 Research article ......................................................................................................................................................................................................................................... 14. 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Submitted on 30 November 2008; accepted on 18 December 2008; advance access publication 16 April 2009 ......................................................................................................................................................................................................................................... http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Bioscience Horizons Oxford University Press

The anti-proliferative effect of different tomato varieties on the human colon adenocarcinoma cells

Saunders, Caroline
Bioscience Horizons , Volume 2 (2) – Jun 16, 2009
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Volume 2 † Number 2 † June 2009 10.1093/biohorizons/hzp020 ......................................................................................................................................................................................................................................... Research article The anti-proliferative effect of different tomato varieties on the human colon adenocarcinoma cells Caroline Saunders* Department of Food Biosciences, School Chemistry, Food and Pharmacy, University of Reading, Reading, UK. * Corresponding author: 47 Beech Lane, Earley, Reading, Berkshire RG6 5PT, UK. Tel: þ44 1189 671022. Email: caroline.saunders50@ntlworld.com Supervisor: Dr Jeremy P.E. Spencer, Molecular Nutrition Group, School Chemistry, Food and Pharmacy, University of Reading, Reading, UK ........................................................................................................................................................................................................................................ Epidemiological evidence suggests that diets rich in fruit and vegetables protect against the development of colon cancer, primarily due to their high levels of polyphenols, in particular a group known as flavonoids. Tomatoes contain significant amounts of the glycosides of the flavonoids quercetin and naringenin. Both quercetin and naringenin have been shown to exert anti-proliferative effects on colon cancer cells, although the effects of whole tomato polyphenol extracts are unclear. The aim of this study was to determine the effect of three tomato varieties, with differing levels of flavonoids and total phenolics, on the proliferation of human colon adenocarci- noma cells. We show that all three varieties were able to significantly inhibit the growth of colon cancer cells, although this activity was found not to be linked to the levels of the flavonoids rutin and naringenin in the tomatoes, but rather to their total phenolic content. We show that the mechanism of growth inhibition was linked to the effects of tomato phenolics on the cell cycle, in that exposure of cells to the tomato extract induced a reduction in the percentage of cells in the S-phase, i.e. those actively synthesizing DNA. These data indicate that tomatoes may help to prevent colon cancer but that their flavonoid content does not appear to determine the magnitude of their biological effect. Key words: colon cancer, tomato, flavonoids, naringenin, rutin. ........................................................................................................................................................................................................................................ and vegetables appear to provide protection against cancer Introduction development due to their levels of fibre and their high con- Colorectal cancer is one of the major cancers of the Western tents of phytochemicals, in particular flavonoids. world with countries such as Australia and New Zealand Flavonoids are secondary plant metabolites, which are having a 20-fold higher incidence than countries in Middle synthesized from phenylalanine via the Shikimate Pathway, Africa. The two major risk factors in colon cancer aetiology and express many functions within the plant, including regu- have been proposed, genetic disposition and diet, although lation of growth and UV protection. The basic structure of the variability between countries appears to result from the flavonoids consists of two benzene rings connected by an latter as migrant populations develop a similar colon oxygen-containing pyrene ring (Fig. 1). On the basis of vari- cancer incidence to that of their new country within a gener- ations in the saturation of the basic flavan ring system, their ation. Indeed, epidemiological evidence recorded between alkylation and/or glycosylation and the hydroxylation 1966 and 1996 has suggested very strong links between pattern of the molecules, flavonoids may be divided into diet and cancer of the breast, colorectum and prostate. seven subclasses: flavonols, flavones, flavanones, flavanonols, One of the reasons that the colon is so susceptible is due to flavanols, anthocyanidins and isoflavones (Fig. 1). Evidence its direct exposure to dietary carcinogens such as N-nitroso indicates that flavonoids express a wide variety of biological 7 6 compounds, such as those present in preserved meats, activities including antioxidant nature, anti-inflammatory which cause DNA damage and lead to mutations and the and anti-aggregatory effects and an ability to inhibit 3 9–12 initiation of carcinogenesis. In contrast, diets high in fruit cancer cell growth. Tomatoes (Lycopersicon ......................................................................................................................................................................................................................................... 2009 The Author(s). This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. 172 Bioscience Horizons † Volume 2 † Number 2 † June 2009 Research article ......................................................................................................................................................................................................................................... Figure 1. Basic structure of flavonoids consisting of two benzene rings connected by an oxygen containing pyrene ring. Sub-classes of flavonoids: flavonols, flavanones, flavanols, anthocyanidins and with differing R groups that give each flavonoid its unique structure. esculentum) are one of the world’s common vegetables and which is produced from naringenin chalcone during tomato contain significant amounts of the flavonol, rutin (quercetin processing. 3-rutinoside) and the flavanone, naringenin and naringenin A number of case–control studies have found an inverse 13, 14 chalcone. Rutin is present in tomato as a glycoside relationship between tomato intake and the risk of develop- 18, 19 and as such undergoes very limited absorption in the small ing colorectal cancer. Furthermore, in vitro and in vivo intestine, as the b-glycosidic bond of the rutinose moiety is studies have demonstrated a wide range of biological activi- resistant to hydrolysis by digestive enzymes. However, ties related to tomato flavonoids, with quercetin and rutin enzymes produced by the colonic gut microflora are able to inhibit abnormal colonic growths in rats and quer- capable of breaking this glycosidic linkage to yield quercetin, cetin chalcone, capable of reducing the size of implanted 9, 16 21 which has been shown to be bioavailable. Flavonoids colon tumours in mice. In humans, quercetin, in combi- that are not well absorbed in the small intestine and the nation with the polyphenol curcumin, was shown to reduce major proportion of ingested flavonoids pass to the large the number and size of ileal and rectal adenomas in patients intestine where they may express biological function on with familial adenomatous polyposis (FAP). Furthermore, host cells and bacteria. This is also true of naringenin quercetin, but not rutin, has been shown to express ......................................................................................................................................................................................................................................... 173 Research article Bioscience Horizons † Volume 2 † Number 2 † June 2009 ......................................................................................................................................................................................................................................... anti-proliferative effects on human colon cancer cell lines, in including a sample selectively bred to be high in polyphenols, 9, 10, 23, 24 a dose-dependent and time-dependent manner. a low flavonoid mutant and a traditional Ailsa Craig variety. 9, 23 Their ability to induce apoptosis and/or evoke cell All samples were stored in the dark at room temperature. 25 –27 cycle arrest have been proposed as potential anti-cancer Polyphenols, hydroxycinnamates, flavonoids and their glyco- mechanisms. The latter has been proposed to occur via their sides were extracted from lyophilized tomato by three ability to regulate cyclins, a family of proteins that are posi- sequential aqueous/methanol extractions. A methanol/ tive regulators of the cell cycle and which are commonly water (2:1) solution was prepared and a total volume of over-expressed in cancer. In response to growth factors 1 ml added to 100 mg lyophilized tomato. Samples were vor- and mitogens, cyclins associate with cyclin-dependent texed continuously for 30 min using a vibrating agitator. kinases (CDKs), which then phosphorylate cellular proteins Samples were then microfuged at 10 000 rpm for 5 min at resulting in the progression of the cell through the cell cycle. 4 C and the supernatants collected. The pellet was A tomato digestate containing both rutin and naringenin re-suspended in 1 ml of the aqueous methanol solution and chalcone has been shown to arrest cell cycle progression at the procedure repeated a further two times. Samples were the G0/G1 phase in colon adenocarcinoma cells through purged with argon and protected from light throughout the decreased expression of cyclin D1, a positive regulator of extraction. the cell cycle. Furthermore, many flavonoids present in tomatoes have been shown to be capable of inhibiting the Quantification of Total Phenolics cell cycle and modulating cyclin expression. For example, The total phenolic content of the aqueous methanolic extract 2 -OH flavanone, a flavonoid with structural similarities to of each tomato sample was obtained using the Folin– naringenin, induces cytotoxic effects on three colorectal car- Ciocalteau Micro method as previously described. cinoma cells via a mechanism dependent on increased Twenty microlitres of the tomato extracts (10 mg/ml) were expression of p21, a potent CDK inhibitor capable of block- mixed with 1.58 ml water and 100 ml of the Folin– ing cells in the G1/S phase of the cell cycle. In addition, Ciocalteu reagent, vortexed for 5 min. Following this, quercetin has been shown to block the cell cycle at the 300 ml of sodium carbonate solution (250 mg/ml) was G1/S phase in human colon cancer cells, by inhibiting the added and the reactants were mixed and left at 4 C for synthesis of a 17 kDa protein. In this study, we have deter- 30 min. Absorbance was determined at 765 nm against the mined the major flavonoid content and total polyphenol blank, and a gallic acid calibration curve (0–500 mg/l) levels of three different tomato varieties and related this to was constructed and used to determine the total phenolic their ability to inhibit the growth of human epithelial color- content of the samples, expressed as Gallic Acid ectal adenocarcinoma cells. In addition, we have attempted Equivalents (GAE). to determine how they act via assessment of their capability to induce cell cycle arrest. Assessment of Flavonoid Levels Reverse phase HPLC (RP-HPLC) was performed to charac- Materials and Methods terize and quantify the flavonoids rutin and naringenin. A Hewlett Packard (Agilent, Bracknell, UK) model 1100 Chemicals and Reagents series LC running HP ChemStation software with a Nova Human epithelial colorectal adenocarcinoma cells (Caco-2) Pak C column (250  4.6; 4 mm) (Waters, Elstree, UK) were obtained from ECACC (Salisbury, Wiltshire, UK). was used to separate the phenolic constituents. The mobile Methanol [high-performance liquid chromatography phase consisted of (A) aqueous methanol (5%) containing (HPLC) grade] and N, N-dimethylformamide (analytical HCl (0.1%) and (B) acetonitrile (50%) containing HCl grade) were purchased from Fisher (Loughborough, UK); (0.1%) and was pumped through the column at 1 ml/min Anti-bromodeoxyuridine (BrdU) Pure was purchased from with an injection volume of 50 ml. The following gradient Becton Dickinson (Oxford, UK); DMEM was purchased system was used over a time course of 60 min (%A/%B): from Cambrex Bio Science (Wokingham, UK). All other 95/5, 50/50, 0/100 and 95/5. HPLC profiles of extracts cell culture components were from Gibco Invitrogen were measured by a diode-array detector set at four wave- (Paisley, UK). All other chemicals were from Sigma-Aldrich lengths: 254, 280, 320 and 365 nm and fluorescence detec- (Gillingham, UK). tion at excitation 276 nm, emission 316 nm. Flavonoids were identified by matching their retention times and UV Tomato Samples and Extraction diode array spectra with those of authentic compounds. Lyophilized tomato samples were supplied by Professor Peter Calibration curves of rutin and naringenin were constructed Bramley, School of Biological Sciences, Royal Holloway, using authentic standards (0–100 mM) and in each case were University of London. All samples were glasshouse grown found to be linear with correlation coefficients of 0.998 under supplementary lighting. Three samples were used, (rutin) and 0.999 (naringenin). ......................................................................................................................................................................................................................................... 174 Bioscience Horizons † Volume 2 † Number 2 † June 2009 Research article ......................................................................................................................................................................................................................................... Cell Culture, Treatment and Assessment of Proliferation Statistics Caco-2 cells were grown as monolayers in T-75 culture flasks All data are expressed as means+ SD. The results of the (Greiner-Bio-One, Frickenhausen, Germany). Cells were cul- Folin–Ciocalteu assay were subjected to a one-way tured in Dulbecco’s modified Eagle’s medium, supplemented ANOVA with Tukey’s post hoc test. Statistical analysis of with 20% heat-inactivated foetal bovine serum, 2 mM Caco-2 proliferation in response to tomato treatments was L-glutamine, 1% non-essential amino acids, 100 U/ml peni- performed using a two-way ANOVA with Dunnet’s post cillin and 100 mg/ml streptomycin and stored in a humidified hoc test. Differences were considered significant when p , 37 C incubator with 5% carbon dioxide. Medium was 0.05. All statistical analyses were performed using SPSS. changed every 2 days and cells were passaged every 7 days using trypsin-versene (EDTA). Prior to the addition of tomato extracts, cells were seeded in 12-well plates (1.2  Results 10 cells/well). When cells were 30% confluent, they were Polyphenolic Content of the Tomato Extracts exposed to the tomato extracts (0.33–3.3 mg). Following 24, 48, 72 and 96 h, cells were fixed and cell biomass was Quantification of phenolics was determined by the Folin– determined using the sulforhodamine B (SRB) assay, as Ciocalteu Assay and statistical analysis using a one-way described previously. Cells were fixed by the addition of ANOVA with Tukey’s post hoc test. Results were expressed 125 ml ice-cold TCA (10% final concentration; 48C; 1 h). as GAE and indicated that the Ailsa Craig variety had a sig- After fixing, media was removed, cells were washed and nificantly higher phenolic content ( p , 0.001) compared total biomass was determined using SRB (250 ml of 0.4% with the selectively bred high-polyphenol variety and the SRB; 0.5 h). Unincorporated dye was discarded by washing low-polyphenol mutant (Fig. 2). However, the high- with 1% acetic acid, while cell incorporated dye was solubil- polyphenol variety had significantly higher levels of total ized using Tris Base (10 mM, pH 10.5). Dye incorporation, phenolics compared with the low-polyphenol mutant ( p , reflecting cell biomass, was measured at 492 nm, using a 0.05) (Fig. 2). GENios microplate reader (TECAN, Reading, UK). Flavonoid Identification and Quantification HPLC analysis (365 nm) indicated that all three tomato var- ieties revealed that three major flavonoids were present, as Cell Cycle Analysis 4 defined by the appearance of peaks at detectable at 31.7, Caco-2 cells were seeded in 12-well plates (1.2  10 cells/ 34.1 and 50.4 min (Fig. 3). The Ailsa Craig variety well) and grown until 30% confluent before exposure to (Fig. 3Ai) had significantly higher levels of rutin (quercetin-3- tomato extracts (1.7 mg/ml; 48 h). After 48 h of exposure, 8 rutinoside) (RT: 34.1 min) and naringenin chalcone (RT: BrdU (10 mM) was added and cells were incubated at 37 C 50.4 min) than either the low mutant (Fig. 3Aii) or high- for 30 min, prior to washing (PBS) and harvesting of cells polyphenol tomatoes (Fig. 3Aiii). The tomatoes also con- (250 ml trypsin/well). Cells were re-suspended in ice-cold 8 tained another, more polar, flavonoid with similar spectral 70% ethanol (1 ml) and stored at 4 C for 24 h. Cells were centrifuged at 2000 rpm (5 min; 4 C), the ethanol removed and the cells re-suspended in HCl (0.1 M; 10 min) in order to permealize them. After washing, cells were incubated for 60 min with a BrdU antibody (5 mg/ml) in PBS containing 0.5% Tween 20, 1% foetal calf serum (FCS). Cells were then washed (PBS) and centrifuged (2000 rpm; 10 min; 4 C) prior to removal of the supernatant and addition of FITC-conjugated rabbit anti-mouse immunoglobulin (0.1 mM) in PBS, containing 0.5% Tween 20 and 1% FCS. This was mixed and incubated in the dark for 30 min. Following a final wash step (PBS) and centrifugation (2000 rpm; 10 min; 4 C), the supernatant was removed and propidium iodide (50 mg/ml) in PBS containing RNase (10 mg/ml) was added. The number of proliferating cells Figure 2. Total phenolic content (expressed as equivalents of gallic acid) was determined by flow cytometry using a FACS Calibur of three different varieties of lyophilized tomatoes determined by the benchtop flow cytometer (Becton-Dickinson, Oxford, UK) Folin–Ciocalteu assay and statistical analysis using a one-way ANOVA. equipped with a 15 mW blue argon laser source (excitation Data are means of three technical replicates of each sample (mean+ SD; wavelength: 488 nm) and were analysed using CellFIT n ¼ 3). A significant difference in total phenolic content between samples is represented by: *p , 0.05 and ***p , 0.001. Cell-Cycle Analysis Version 2.0.2 software. ......................................................................................................................................................................................................................................... 175 Research article Bioscience Horizons † Volume 2 † Number 2 † June 2009 ......................................................................................................................................................................................................................................... Figure 3. (A) RP-HPLC chromatogram recorded at 365 nm of tomato extracts (concentration 10 mg/ml), (i) Ailsa Craig; (ii) low polyphenol and (iii) high polyphenol. (B) Content of flavonoids, rutin and naringenin derivatives, in tomatoes extracted with aqueous methanol (2:1) and expressed as mg/glyo- philized tomato. Quantified using ‘area-under-curve’ analysis relative to an internal standard and by comparison with calibration curves of rutin and naringenin. characteristics to rutin (RT: 31.7) (Fig. 3). Although we were product. In quantitative terms, the Ailsa Craig variety had unable to characterize this compound, it is likely to be either a much higher levels of both rutin and naringenin chalcone more polar glycosidic derivative of quercetin or a myricetin compared with the low mutant or high-polyphenol varieties glycoside (Fig. 1). LC-MS/MS is underway to identify this (Fig. 3B). Although our extraction and separation protocol ......................................................................................................................................................................................................................................... 176 Bioscience Horizons † Volume 2 † Number 2 † June 2009 Research article ......................................................................................................................................................................................................................................... is capable of revealing the hydroxycinnamates, all three tomatoes were found not to contain significant levels (Fig. 3). Effect on Colon Adenocarcinoma Cell Proliferation The cytotoxic effects of the three tomato extracts were indi- cated by a reduction in cell biomass compared with vehicle- treated cells which was determined by the SRB assay. A stat- istically significant ( p , 0.05) inhibition of cell growth was apparent following exposure to Ailsa Craig (Fig. 4A) and high polyphenol (Fig. 4B) at both concentrations and at both time points and the low polyphenol (Fig. 4C) after 72 h exposure only. At the 72 h exposure time point, there was a statistically significant difference in the ability of the Ailsa Craig extract to inhibit cell proliferation compared with the low-polyphenol sample ( p , 0.05) (Fig. 5). Cells exposed to the high-polyphenol tomato extract (1.7 mg/ Figure 5. Comparison of growth inhibitory effect of the three different ml) and vehicle-treated cells were analysed by flow cytometry tomato varieties, on proliferation of Caco-2 cells, relative to vehicle-treated cells, after 72 h of exposure to sample. Values are given as means+ SD (n ¼ 4). A significant difference of growth inhibition between samples is represented by: *p , 0.05. for the number of cells actively synthesizing DNA (i.e. in the S-phase of the cell cycle) (Fig. 6). Emission spectra of FITC indicated a shift in cell cycle distribution in cells exposed to the high-polyphenol extract, as indicated by a decreased cellular incorporation of BrdU (Fig. 6). Discussion A number of case–control studies have shown an inverse relationship between tomato intake and the risk of developing 16, 17 colorectal cancer. Furthermore, in vitro and animal studies have shown that tomatoes, and phenolic compounds present in tomatoes, are able to inhibit colon cell prolifer- ation. In this study, we demonstrate that three different tomato extracts are capable of significantly inhibiting colon adenocarcinoma cell growth. However, this ability to effect growth did not appear to be dependent on the flavonoid content of the tomatoes. The most abundant flavonoids in the tomatoes we studied were the flavonol glycoside, quercetin 3-rutinoside (also known as rutin) and the flavanone, narin- 13, 14 genin chalcone. Interestingly, although the high- polyphenol variety had been selectively bred to have an increased content of polyphenols, the traditional Ailsa Craig variety had higher levels of total polyphenols and .10 times the rutin and naringenin chalcone levels than this tomato. The low-polyphenol mutant as expected had lower levels of Figure 4. Growth inhibition of Caco-2 cells induced by each methanolic total phenolics compared with both other varieties and also tomato extract treatment. Black bars: 0.5 mg/ml and grey bars: 2.5 mg/ had very low levels of rutin and undetectable levels of narin- ml, after 48 and 72 h initial exposure. (A) Ailsa Craig (B) high polyphenol genin chalcone. Although all the tomatoes induced anti- and (C) low polyphenol. All values are given as means+ SD (n ¼ 4). A sig- proliferative effects, the degree of growth inhibition (Fig. 5) nificant difference of growth inhibition at an individual time point and con- appeared to be dependent on the total phenolic content of centration, compared with vehicle-treated cells, is represented by: *p , 0.05; **p , 0.01 and ***p , 0.001. the tomatoes (Fig. 2) rather than their levels of rutin and ......................................................................................................................................................................................................................................... 177 Research article Bioscience Horizons † Volume 2 † Number 2 † June 2009 ......................................................................................................................................................................................................................................... Figure 6. Effect of high-polyphenol methanolic tomato extract on active DNA synthesis determined by the incorporation of BrdU into cell DNA during S-phase of cell cycle. Analysis was by flow cytometry. Black line: vehicle-treated cells; red line: high-polyphenol extract showing a decrease in cells in DNA synthesis phase (S-phase) of the cell cycle (n ¼ 1). naringenin (Fig. 3B). For example, the large difference in levels distribution of cells in the S-phase of the cell cycle. Cancer of rutin and naringenin in the Alisa Craig variety compared cells treated with the high-polyphenol tomato extract under- with the other two tomatoes was not reflected in their ability went a reduction in proliferation which was accompanied by to influence cancer cell growth inhibition. a reduction in the number of cells actively synthesizing DNA, This is in agreement with previous studies which suggest relative to vehicle-treated cells. This is in agreement with a that the flavonol glycoside, rutin, is not able to induce anti- previous study which showed the ability of a tomato diges- proliferative effects until the glycosidic bond of the rutinose tate to inhibit growth of HCT-116 colon adenocarcinoma moiety is cleaved to yield quercetin, which has previously cells was mediated by its ability to block the cell cycle in been shown to exert strong anti-proliferative effects. G0/G1. This block was accompanied by down-regulation Such cleavage may occur in the large intestine via the of cyclin D1, a positive regulator of the cell cycle. action of the colonic gut microflora, indicating that Furthermore, polyphenols in olive oil have been shown to although rutin is incapable of preventing the growth of inhibit proliferation of Caco-2 cells by inducing a cell cycle cancer cells in vitro, it may still be able to induce positive block in G2/M. This block was induced via the upstream reductions in the growth of cancer cells in vivo. The corre- inhibition of the MAP kinase, p38 and the transcription lation between total phenolic content and the degree of inhi- factor, CREB. Further work is required in order to determine bition of colon adenocarcinoma cell growth suggests that the precise mechanism by which tomato polyphenols induce other polyphenolic compounds, or the synergistic effect of the anti-proliferative effects in cells, although our initial a number of different polyphenols, are likely to be respon- observations suggest that they are also able to prevent cell sible for the observed growth inhibition. As our extraction cycle progression. and analysis protocol was capable of detecting and quanti- In summary, the polyphenol extracts of all three tomato fying hydroxycinnamates, our data also suggest that this varieties were able to inhibit the growth of colon adenocar- group of polyphenols is also unlikely to be responsible for cinoma cells and the growth inhibition did not appear to be the anti-proliferative effects as all the tomatoes contained due to their flavonoid or hydroxycinnamate content. The very low levels. Further experiments are necessary in order growth inhibitory effect was accompanied by a shift in cell to identify and characterize the polyphenols in the tomatoes cycle distribution, suggesting that fewer cells were present which are responsible for their biological actions on cancer in the S-phase of the cell cycle following treatment. Future cells. studies are needed in order to identify the specific com- Our data suggest that the mechanism by which the high- ponent(s) present in tomatoes which are responsible for the polyphenol tomato extract exerts its anti-proliferative growth inhibitory effects and to determine the mechanism effects is linked to its ability to induce a shift in the by which they induce their effect on the cell cycle. ......................................................................................................................................................................................................................................... 178 Bioscience Horizons † Volume 2 † Number 2 † June 2009 Research article ......................................................................................................................................................................................................................................... 14. 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Slimestad R, Verheul MJ (2005) Content of chalconaringenin and chloro- and COX-2 expression by olive oil polyphenols underlies their anti- genic acid in cherry tomatoes is strongly reduced during postharvest ripen- proliferative effects. Biochem Biophys Res Commun 362: 606–611. ing. J Agric Food Chem 53: 7251–7256. Author Biography Caroline Saunders completed a BSc in Nutrition and Food Science at the University of Reading and is now about to undertake a PhD at Reading on the subject of flavonoids, genotype and cognitive function. Her undergraduate project allowed her to gain valuable experience and an insight into the field of research which helped make the decision to continue to do a PhD. ........................................................................................................................................................................................................................................ Submitted on 30 November 2008; accepted on 18 December 2008; advance access publication 16 April 2009 .........................................................................................................................................................................................................................................

Journal

Bioscience Horizons – Oxford University Press

Published: Jun 16, 2009

Keywords: Key words colon cancer tomato flavonoids naringenin rutin

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